Hereditary nonspherocytic hemolytic disease associated with an altered phospholipid composition of the erythrocytes.

A hemolytic disorder with mild hyperbilirubinemia and reticulocytosis of 6 to 15% was documented in eight members of a large family from the Dominican Republic and was presumed to be present in eight other members. The disorder appeared to be inherited as an autosomal dominant characteristic. Analysis of phospholipids by quantitative thinlayer chromatography revealed a distinct increase in phosphatidyl choline (lecithin) to 35.5 +/- SD 1.3% of the total (normal: 28.2 +/- 1.4%) in erythrocytes of affected members of the family, but not in the cells of unaffected relatives. The alteration appeared to constitute an absolute increase in lecithin content, rather than a decrease in other phospholipids. Erythrocytes from patients with other varieties of hereditary hemolytic disorders and comparable levels of reticulocytosis had normal phospholipid compositions. Plasma lipids of six affected members of the family were not unusual with respect to total lipid weight, total phospholipid, and cholesterol. Three patients with liver disease and jaundice were found to have marked increases in the lecithin content of the erythrocytes, but they also had extremely high plasma levels of total lipid, phospholipids, and cholesterol. Osmotic fragility of the erythrocytes of affected patients was decreased and the increase in fragility after incubation for 24 hr was less than that observed with normal erythrocytes. Autohemolysis after 48 hr was slightly increased and was corrected to nearly normal by the addition of glucose. The activities of 15 enzymes of the erythrocytes of the propositus were normal or elevated and the adenosine triphosphate content was normal. An abnormal hemoglobin could not be demonstrated. The life span of isologous erythrocytes in the propositus was reduced, but homologous erythrocytes survived normally.A causal relationship between the altered phospholipid composition and the hemolytic disorder has not been established.

The effects of inaccurate blood sample volume on prothrombin time (PT) and activated partial thromboplastin time (aPTT).

The results of determinations of the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are frequently used to assess hemostatic function. Accurate results for these laboratory tests depend on many variables, one of which is the ratio of plasma to anticoagulant. We studied 12 patients and 4 normal subjects to determine the effects of sample volume on PT and aPTT. We conclude that underfilling may produce profound effects, particularly on the aPTT. In contrast, overfilling rarely affects the results. The greatest effects of sample volume were observed in specimens in which the true PT or aPTT was elevated. A normal PT or aPTT result on any specimen, regardless of sample volume, strongly suggests that the true value is normal.

The diagnostic significance of a prolonged erythrocytic glycerol lysis time (GLT50).

The predictive value of a prolonged glycerol lysis time (GLT50) was assessed by analysis of case records of 100 consecutive subjects with values greater than 73 seconds (normal = 26--73 seconds) reported by the clinical laboratory of The New York Hospital. There were 72 cases of hemoglobinopathy: 65 thalassemia trait, four sickle-thalassemia, and one each of Hb D-thalassemia, sickle-C disease, and sickle-cell anemia. Nine of the remaining subjects had iron-deficiency anemia, three had chronic renal disease, and seven had miscellaneous disorders. Four subjects were apparently normal, and in five cases there was insufficient information for a diagnosis. Of 78 patients who had both a prolonged GLT50 and microcytosis, 67 (86%) had thalassemia trait and seven (9%) had iron-deficiency anemia. In 74 patients with GLT50 greater than 100 seconds, thalassemia trait was found 16 times as often as uncomplicated iron-deficiency anemia. All 31 subjects with GLT50 greater than 180 seconds had hemoglobinopahy. A prolonged GLT50 strongly suggests thalassemia trait, especially when greater than 100 seconds or associated with microcytosis.

Prothrombin time and activated partial thromboplastin time can be performed on the first tube.

The current practice standard for coagulation studies on venous blood specifies the use of the second (or subsequent) tube for testing. Unless other tests are ordered, the first tube is discarded. This practice, derived from early experience using nondisposable materials, gained support from a report based on modern phlebotomy methods but inexperienced phlebotomists. For our study, paired blood specimens were collected by experienced phlebotomists from 175 outpatients whose physicians had ordered coagulation tests. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were performed on the first and second tubes. Similar values were obtained for PT and APTT. (PT: n = 174; mean = 15.36 seconds; mean difference = 0.10 seconds; y = 1.02x - 0.28; t = 0.24; P>.81; r = 0.995; APTT: n = 160; mean = 38.25 seconds; mean difference = 0.48 seconds; y = 1.03x - 0.71; t = 0.25; P=.80; r = 0.993.) For coagulation tests on specimens collected by experienced phlebotomists, the first tube yields accurate results and need not be discarded.

Utility of the three-part leukocyte differential count.

The utility of the automated three-part leukocyte differential count was evaluated in an acute care hospital setting. The manual method was specified in 92 of 223 (41%) requests for differentials on one day. For the others, 79 three-part differentials (60%) were completed; 19 (15%) were rejected for histogram abnormalities ("R-flags") or could not be computed; and 33 (25%) were rejected for out-of-range values that were later verified by slide review. The automated method missed 4 of 39 (10%) band elevations and 2 of 2 (100%) cases of eosinophilia reported on manual differentials, but those results had no apparent influence on patient management. In 98 cases of septicemia, automated and manual methods showed similar overall sensitivity (87% and 83%, respectively). Selectively combined with a qualitative slide review, the three-part differential was applicable to 84% of all specimens submitted for a differential count, with acceptable sensitivity and accuracy and substantial savings in personnel time.

Laboratory medicine education in United States medical schools.

The Academy of Clinical Laboratory Physicians and Scientists conducted a survey of US medical schools to examine the current status of laboratory medicine education, estimate the amount of teaching time available in the curriculum, and identify the most effective teaching practices. Questionnaires were sent to department heads and course directors at 126 US medical schools. Replies were received from 120 schools (95%), 83 of which offered a total of 132 courses in laboratory medicine. Only 68 schools (57%) had required courses. Most of the elective courses (35 of 50; 70%) were in general clinical pathology. Lectures remained the most common teaching format, with or without laboratory sessions and workshops. Computer-assisted instruction was used in only 10 schools. Laboratory medicine courses were offered in all 4 years of medical school, with the majority (70 of 132; 53%) in the second year, often integrated with general pathology. Opinion was divided over the relative importance of laboratory medicine instruction in the preclinical versus clinical years.

Reliable estimation of hemoglobin A2 concentration by electrophoresis with densitometry.

Elevated hemoglobin A2 (Hb A2) levels can be identified conveniently by densitometry after electrophoresis on cellulose acetate strips. Because a recent report questioned the accuracy of this technic, the method was re-evaluated by paired comparison with microcolumn chromatography. Analysis of 100 patient specimens showed high correlation (r = +0.84), but an average Hb A2 concentration 0.7% higher by densitometry than by chromatography (P less than 0.001). With upper limits set at 4.5%, and 3.8%, respectively, results were divided into "normal" and "high" for each method. Concordant results were obtained in 97 of the 100 cases (82 normal, 15 high). Another densitometer of improved design was used for paired analysis of 50 additional specimens, 25 normal and 25 with beta-thalassemia trait. The two groups were well separated by both procedures, and Hb A2 levels were similar (r = +0.92, P greater than 0.6). This study demonstrates that it is possible, with carefully controlled technics and properly calibrated instruments, to use electrophoresis with densitometry as a reliable means of identifying abnormal Hb A2 levels.

Electronic platelet counts with the Coulter counter. Reassessment of a correction factor.

Platelet counts are determined on the Coulter electronic counter by counting the diluted platelet-rich plasma obtained by sedimentation or centrifugation of whole blood. In calculating the whole-blood platelet count, an empirical correction factor for platelet-free plasma trapped by sedimented erythrocytes has been recommended, and a widely-distributed circular slide rule calculator incorporates the correction factor. In this study, visual and electronic platelet counts were compared in 100 specimens with counts ranging from 10 to 1,100 X 10(3) per mul and hematocrits ranging from 17.5 to 48.5%. Platelet-rich plasma samples prepared by a centrifugation method (Plateletfuge) gave machine counts in close agreement with those of samples prepared by sedimentation. Whole-blood platelet counts determined with the circular calculator were consistently lower than visual counts, with an average difference of -17%. The electronic counts were recalculated after elimination of the correction factor, and agreement then improved to an average difference of only +1.6%. The correction factor for trapped platelet-free plasma leads to erroneously low values and should not be used.

Failure of the International Normalized Ratio to generate consistent results within a local medical community.

Use of the International Normalized Ratio (INR) has been recommended as a means of standardizing prothrombin time (PT) results for management of oral anticoagulant therapy. During the evaluation of a new lot of thromboplastin reagent, however, INR values were obtained that were inconsistent with results obtained with the prior lot of reagent from the same manufacturer. A local normalized ratio (LNR) was substituted for the INR for the new reagent, based on a calculated local sensitivity index (LSI). Validation of the LSI was performed at the three hospitals in the medical community by testing an identical panel of aliquots from 64 plasmas obtained from patients who were chronically receiving oral anticoagulants. Each test result was classified according to the INR value as low, low therapeutic, high therapeutic, or high. Local normalized ratio values obtained at one hospital in the community were in reasonable agreement with INR determinations in the other two hospital laboratories. Classification mismatches occurred using the INR, however, in 84 of 280 (30%) of the paired samples. Thus, the inability to generate consistent INR values within a local medical community raises serious concern about the reliability of the INR concept in its current form.

Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant.

Underfilling of specimen tubes containing 129 mmol/L (3.8%) buffered citrate prolongs prothrombin time (PT) and activated partial thromboplastin time (APTT) values. We studied this phenomenon by using 109 mmol/L (3.2%) buffered citrate as the anticoagulant, anticipating some increase in tolerance to underfilling. Venous blood drawn from 12 healthy subjects and 30 patients receiving long-term oral warfarin therapy was mixed with 109 mmol/L buffered citrate solution in proportions equivalent to filling the collection tubes from 52% to 100% of capacity. Accurate PT values were obtained from normal specimens if the tubes were filled to 65% or more of capacity. Accurate PT results in the therapeutic range were obtained only with filling to 80% or more of capacity (using a "moderately sensitive" thromboplastin reagent, International Sensitivity Index [ISI] = 2.06) or 90% or more of capacity (using a "highly sensitive" thromboplastin reagent, ISI = 1.01). In contrast, APTT was much less tolerant to underfilling, with prolonged values observed in most specimens filled to less than 90% of capacity. No false low values were observed. Specimen tubes should be filled to at least 90% of capacity to avoid falsely elevated PT or APTT results, but values within the reference range may be acceptable even from underfilled tubes.

Highly sensitive thromboplastins do not improve INR precision.

For determination of the international normalized ratio (INR), it has been suggested that "highly sensitive" thromboplastin reagents (International Sensitivity Index [ISI] < or = 1.2) provide the most consistent performance and minimize interlaboratory variability. We compared the INR values obtained from 69 specimens drawn from patients receiving long-term oral anticoagulant therapy, using four thromboplastin preparations (manufacturer-assigned ISI range of 0.96-1.10) and two automated photo-optical analyzers. Multivariate analysis of the INR response matrix (552 INR values) indicated that the eight reagent-coagulometer combinations did not produce equivalent INR values. Similar analysis indicated that INR values were not normalized when uncorrected prothrombin ratios or INR values, calculated after assignment of "local ISI values" to each thromboplastin reagent, were compared. The INR differences also seemed to be clinically significant because 17% to 29% of paired thromboplastin values were discordant when all INR values were assigned to one of four therapeutic categories used in oral anticoagulant therapy (< 2.0; 2.0-3.0; 3.0-4.5; or > 4.5). These differences in INR values obtained with two photo-optical coagulometers and four highly sensitive thromboplastin reagents suggest that the existing INR system has not achieved the goal of standardized prothrombin time values and does not support the recommendation to use only highly sensitive reagents for the regulation of oral anticoagulant therapy.

Case report. Clostridium perfringens septicemia with detection of phospholipase C activity in the serum.

Clostridium perfringens septicemia with massive hemolysis is described in an elderly woman with refractory anemia. This case is highly unusual in that (1) the diagnosis was made during life by discovery of the organisms on a routine peripheral blood film, and that (2) phospholipase C activity was detected in the serum.

Lipid patterns in human leukocytes maintained in long-term culture.

The lipid composition of leukocytes maintained in long-term culture was examined in order to clarify the role of immaturity in previously observed differences between normal mature leukocytes and leukemic cells. Cell cultures derived from three types of leukocytes were examined: normal lymphocytes, Burkitt lymphoma, and chronic myelocytic leukemia. Lipid extracts were analyzed for total lipid weight, phospholipids, neutral lipids, and glycolipids. Distribution of individual phospholipids was determined by quantitative two-dimensional thin-layer chromatography. The main phospholipids were phosphatidylcholine (51-54%) and phosphatidylethanolamine (24-25%), with smaller amounts of phosphatidylinositol, phosphatidylserine, sphingomyelin, and cardiolipin. All three types of cultured cells showed a remarkable similarity in total phospholipid content (17-18 x 10(-15) moles/cell) as well as in phospholipid distribution. More variation was seen in neutral lipid content. Glycolipid was abundant (17-23% of total lipid weight) and was present mostly as ceramide dihexoside. Compared with normal lymphocytes or polymorphonuclear leukocytes, the cultured cells showed increased phosphatidylcholine, decreased sphingomyelin, and decreased cholesterol content, similar to the changes found in leukemic leukocytes. These findings suggest that the altered lipid patterns found in leukemic leukocytes are a reflection of cell immaturity rather than a characteristic peculiar to the leukemic state.

Lipids of human leukocytes: relation to celltype.

Significant differences in lipid composition have been found between normal human lymphocytes and polymorphonuclear leukocytes (isolated from blood by means of glass-bead columns), abnormal leukocytes from patients with acute and chronic leukemia, and leukocytes from peritoneal exudates. Lipid extracts of isolated leukocytes were analyzed for total lipid, phosphorus, cholesterol, and plasmalogens. Individual phospholipids and neutral lipids were separated by thin-layer chromatography. The major phospholipids were phosphatidyl choline, ethanolamine glycerophosphatides, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol. Plasmalogen was found mainly as phosphatidal ethanolamine. The neutral lipid fractions contained free cholesterol and various amounts of triglyceride, but little esterified cholesterol. Normal lymphocytes contained about half as much total lipid per cell as normal polymorphonuclear leukocytes, with a similar cholesterol:-lipid-P ratio but relatively more lecithin and less ethanolamine glycerophosphatide. Normal mature leukocytes, compared with immature cells of the same morphological series, had a higher total lipid content per cell, more cholesterol, and a higher ratio of cholesterol to lipid-P. Little difference was found in total lipid-P per cell, but mature cells contained relatively less lecithin and more sphingomyelin. These findings may reflect differences in the relative content of various intracellular organelles as well as possible differences in the quantity and composition of the plasma membrane.